Hypertension affects 25 million Americans, contributing importantly to stroke, myocardial infarction and kidney failure. Both environmental and genetic factors contribute to hypertension. The kidney plays a central role in blood pressure homeostasis by controlling salt excretion. An important part of the salt homeostatic system is the thiazide-sensitive Na-CI cotransplorter. Dysfunction of this transport protein leads to Gitelman's syndrome, an autosomal recessive disorder of salt wasting, hypokalemia and alkalosis. We have shown that many mutations causing Gitelman's syndrome create misfolded proteins, thus activating the quality control mechanism of the endoplasmic reticulum. Recently, a hypertensive disorder, pseudohypoaldosteronism type II, has been shown to result from mutations in WNK (without lysine) kinases. We show preliminary data indicating that WNK kinases regulate thiazide-sensitive Na-CI cotransporter activity. WNK4 suppresses thiazide-sensitive Na-CI cotransporter activity by more than 80%. WNK1 does not alter Na-CI cotransporter activity by itself, but instead inhibits WNK4 activity. WNK1 is known to be regulated by osmolality, as is the thiazide-sensitive Na-CI cotransporter. We postulate that the WNK kinase system regulates thiazide-sensitive Na-CI cotransport in response to changes in luminal osmolality. We will test the hypotheses that 1) WNK4 downregulates thiazide-sensitive Na-CI cotransporter activity by interacting with this transport protein and phosphorylating it, 2) WNK1 inhibits WNK4-mediated suppression of thiazide-sensitive Na-CI cotransporter activity by interacting with and phosphoryiating WNK4, 3) pseudohypoaldosteronism type II results from increased WNK1 activity leading to inhibited WNK4 mediated suppression of Na-CI reabsorption by the distal tubule, 4) specific threonine moieties on the Na-CI cotransporter are phosphorylated in response to WNK stimulation, 5) WNK4 is expressed at tight junctions along the distal tubule and regulates paracellular ion permeability, and 6) WNK kinases link paracellular to transcellular conductance. The results of experiments described in this proposal will illuminate molecular mechanisms of hypertension. Although Mendelian forms of hypertension are rare, they help to elucidate mechanisms of blood pressure regulation that almost certainly contribute to the more common essential variety.